Insect Resistant Cotton Plants And Methods For Identifying Same

ABSTRACT

The invention provides specific transgenic cotton plants, plant material and seeds, characterized in that these products harbor a specific transformation event at a specific location in the cotton genome. Tools are also provided which allow rapid and unequivocal identification of the event in biological samples.

FIELD OF THE INVENTION

This invention relates to transgenic cotton plants, plant material andseeds, characterized by harboring a specific transformation event,particularly by the presence of a gene encoding a protein that confersinsect resistance, at a specific location in the cotton genome. Thecotton plants of the invention combine the insect resistance phenotypewith an agronomic performance, genetic stability and adaptability todifferent genetic backgrounds equivalent to the non-transformed cottonline in the absence of insects. This invention further provides methodsand kits for identifying the presence of plant material comprisingspecifically transformation event EE-GH5 in biological samples.

BACKGROUND OF THE INVENTION

The phenotypic expression of a transgene in a plant is determined bothby the structure of the gene itself and by its location in the plantgenome. At the same time the presence of the transgene (in a foreignDNA) at different locations in the genome will influence the overallphenotype of the plant in different ways. The agronomically orindustrially successful introduction of a commercially interesting traitin a plant by genetic manipulation can be a lengthy procedure dependenton different factors. The actual transformation and regeneration ofgenetically transformed plants are only the first in a series ofselection steps, which include extensive genetic characterization,breeding, and evaluation in field trials, eventually leading to theselection of an elite event.

Cotton fiber is the single most important textile worldwide. About 80million acres of cotton are harvested annually across the globe. Cottonis the fifth largest crop in the U.S. in terms of acreage production,with over 15 million acres planted in 2000.

The most damaging insect species feeding on cotton are Helicoverpa zea(corn earworm or cotton bollworm), Helicoverpa armigera (Americanbollworm) Heliothis virescens (tobacco budworm) and Helicoverpapunctigera.

The unequivocal identification of an elite event is becomingincreasingly important in view of discussions on Novel Food/Feed,segregation of GMO and non-GMO products and the identification ofproprietary material. Ideally, such identification method is both quickand simple, without the need for an extensive laboratory set-up.Furthermore, the method should provide results that allow unequivocaldetermination of the elite event without expert interpretation, butwhich hold up under expert scrutiny if necessary. Specific tools for usein the identification of elite event EE-GH5 in biological samples aredescribed herein.

EE-GH5 has been identified as an elite event from a population oftransgenic cotton plants in the development of insect resistant cotton(Gossypium hirsutum) comprising a gene coding for a insecticidal crystalprotein from Bacillus thuringiensis. The transgenic cotton plantscontain a chimeric gene encoding a Bt insecticidal crystal protein (asdescribed in WO03/093484) under control of a plant-expressible promoter.

Cotton plants comprising a insect resistance gene have been disclosed inthe art. However, none of the prior art disclosures teach or suggest thepresent invention.

SUMMARY OF THE INVENTION

The present invention relates to a transgenic cotton plant, or seed,cells or tissues thereof, comprising, stably integrated into its genome,an expression cassette which comprises a insect resistance genecomprising the coding sequence of the cry1Ab gene (as described inExample 1.1 herein), which is insect resistant and, in the absence ofinsect pressure, has an agronomic performance which is substantiallyequivalent to the non-transgenic isogenic line. Under insect pressure,the plant will have a superior agronomic phenotype compared to thenon-transgenic plant.

According to the present invention the cotton plant or seed, cells ortissues thereof comprise elite event EE-GH5.

More specifically, the present invention relates to a transgenic cottonplant, seed, cells or tissues thereof, the genomic DNA of which ischaracterized by the fact that, when analyzed in a PCR identificationprotocol as described herein, using two primers directed to the 5′ or 3′flanking region of EE-GH5 and the foreign DNA, respectively, yields afragment which is specific for EE-GH5. The primers may be directedagainst the 5′ flanking region within SEQ ID NO: 1 and the foreign DNArespectively such as the primers comprising or consisting of thenucleotide sequence of SEQ ID NO: 7 and SEQ ID NO: 8 respectively, andyield a DNA fragment of between 100 and 700 bp, preferably of about 129bp. The primers may also be directed against the 3′ flanking regionwithin SEQ ID NO: 2 and the foreign DNA respectively such as the primerscomprising or consisting of the nucleotide sequence of SEQ ID NO: 3 andSEQ ID NO: 6 respectively, and yield a DNA fragment of between 300 and700 bp, preferably of about 369 bp.

Reference seed comprising the elite event of the invention has beendeposited at the ATCC under accession number PTA-8171. One embodiment ofthe invention is the seed comprising elite event EE-GH5 deposited asATTC accession number PTA-8171, which will grow into a cotton plantresistant to insects, particularly Helicoverpa sp. or Heliothis sp. Theseed of ATCC deposit number PTA-8171, is a seed lot consisting of atleast about 95% transgenic kernels homozygous for the transgene,comprising the elite event of the invention, which will grow into insectresistant plants, whereby the plants are also glufosinate tolerantplants. The seed can be sown and the growing plants can be treated withglufosinate as described herein to obtain 100% glufosinate tolerantplants, comprising the elite event of the invention. The inventionfurther relates to cells, tissues, progeny, and descendants from a plantcomprising the elite event of the invention grown from the seeddeposited at the ATCC having accession number PTA-8171. The inventionfurther relates to plants obtainable by propagation of and/or breedingwith a cotton plant comprising the elite event of the invention grownfrom the seed deposited at the ATCC having accession number PTA-8171.The invention also relates to cotton plants comprising elite eventEE-GH5.

The invention further relates to a method for identifying a transgenicplant, or cells or tissues thereof, comprising elite event EE-GH5 whichmethod is based on identifying the presence of characterizing DNAsequences or amino acids encoded by such DNA sequences in the transgenicplant, cells or tissues. According to a preferred embodiment of theinvention, such characterizing DNA sequences are sequences of 15 bp,preferably 20 bp, most preferably 30 bp or more which comprise theinsertion site of the event, i.e. both a part foreign DNA and a part ofthe cotton genome (either the 5′ or 3′ flanking region) contiguoustherewith, allowing specific identification of the elite event.

The present invention further relates to methods for identifying eliteevent EE-GH5 in biological samples, which methods are based on primersor probes which specifically recognize the 5′ and/or 3′ flankingsequence of EE-GH5.

More specifically, the invention relates to a method comprising ofamplifying a sequence of a nucleic acid present in biological samples,using a polymerase chain reaction with at least two primers, one ofwhich recognizes the 5′ or 3′ flanking region of EE-GH5, the other whichrecognizes a sequence within the foreign DNA, preferably to obtain a DNAfragment of between 100 and 500 bp. The primers may recognize a sequencewithin the 5′ flanking region of EE-GH5 (SEQ ID No. 1, from position 1to position 98 or SEQ ID No. 16 from position 1 to 563) or within the 3′flanking region of EE-GH5 (complement of SEQ ID No 2 from position 41 toposition 452) and a sequence within the foreign DNA (complement of SEQID No 1 from position 99 to 334 or SEQ ID No 2 from position 1 toposition 40), respectively. The primer recognizing the 5′flanking regionmay comprise the nucleotide sequence of SEQ ID No. 7 or SEQ ID No. 17and the primer recognizing a sequence within the foreign DNA maycomprise the nucleotide sequence of SEQ ID No. 8, SEQ ID No. 18 or SEQID No. 19 described herein. The primer recognizing the 3′flanking regionmay comprise the nucleotide sequence of SEQ ID No. 6 and the primerrecognizing a sequence within the foreign DNA may comprise thenucleotide sequence of SEQ ID No. 3 described herein

The present invention more specifically relates to a method foridentifying elite event EE-GH5 in biological samples, which methodcomprises amplifying a sequence of a nucleic acid present in abiological sample, using a polymerase chain reaction with two primershaving the nucleotide sequence of SEQ ID No. 3 and SEQ ID No. 6respectively, to obtain a DNA fragment of about 369 bp.

The current elite event comprises a foreign DNA sequence wherein aslight rearrangement has occurred when compared with the DNA initiallyused for the transformation procedure, the rearrangement being uniquefor elite event EE-GH5. Accordingly, the invention also relates to amethod for identifying elite event EE-GH5 in biological samples, whichmethod comprises amplifying a sequence of a nucleic acid present in abiological sample, using a polymerase chain reaction with two primersflanking the unique rearrangement of foreign DNA in EE-GH5, such as theprimers comprising or having the nucleotide sequence of SEQ ID No 10 andSEQ ID No 11, to obtain a DNA fragment of about 262 bp.

The present invention further relates to the specific flanking sequencesof EE-GH5 described herein, which can be used to develop specificidentification methods for EE-GH5 in biological samples. Such specificflanking sequences may also be used as reference control material inidentification assays. More particularly, the invention relates to the5′ and or 3′ flanking regions of EE-GH5 which can be used for thedevelopment of specific primers and probes as further described herein.The invention also relates to nucleic acid molecules spanning thespecific rearrangements within the foreign DNA of EE-GH5, such as anucleic acid molecule comprising the sequence of SEQ ID No. 12 fromnucleotide position 52 to nucleotide position 88, or comprising thesequence of SEQ ID No.12. Also suitable as reference material arenucleic acid molecules, preferably of about 369 bp, comprising thesequence which can be amplified by primers having the nucleotidesequence of SEQ ID No. 3 and SEQ ID No. 6

The invention further relates to identification methods for the presenceof EE-GH5 in biological samples based on the use of such specificprimers or probes. Primers may consist of a nucleotide sequence of 17 toabout 200 consecutive nucleotides selected from the nucleotide sequenceof SEQ ID No 1 from nucleotide 1 to nucleotide 98 of the nucleotidesequence of SEQ ID No. 16 from nucleotide 1 to nucleotide 563 or thecomplement of the nucleotide sequence of SEQ ID 2 from nucleotide 41 tonucleotide 452) combined with primers consisting of a nucleotidesequence of 17 to about 200 consecutive nucleotides selected from thecomplement of the nucleotide sequence of SEQ ID No 1 from nucleotide 99to nucleotide 334 or the nucleotide sequence of SEQ ID No 2 fromnucleotide 1 to nucleotide 40. Primers may also comprise thesenucleotide sequences located at their extreme 3′ end, and furthercomprise unrelated sequences or sequences derived from the mentionednucleotide sequences, but comprising mismatches.

The invention further relates to kits for identifying elite event EE-GH5in biological samples, said kits comprising at least one primer or probewhich specifically recognizes the 5′ or 3′ flanking region of EE-GH5 orthe specific rearrangement within the foreign DNA sequence in EE-GH5.

The kit of the invention may comprise, in addition to a primer whichspecifically recognizes the 5′ or 3′ flanking region of EE-GH5, a secondprimer which specifically recognizes a sequence within the foreign DNAof EE-GH5, for use in a PCR identification protocol. The kits of theinvention may comprise at least two specific primers, one of whichrecognizes a sequence within the 5′ flanking region of EE-GH5, and theother which recognizes a sequence within the foreign DNA. The primerrecognizing the 5′flanking region may comprises the nucleotide sequenceof SEQ ID No. 3 and the primer recognizing the transgene may comprisesthe nucleotide sequence of SEQ ID No. 6 or any other primer as describedherein.

The invention further relates to a kit for identifying elite eventEE-GH5 in biological samples, said kit comprising the PCR primers havingthe nucleotide sequence of SEQ ID No. 3 and SEQ ID No. 6 for use in theEE-GH5 PCR identification protocol described herein. The inventionfurther relates to a kit for identifying elite event EE-GH5 inbiological samples, said kit comprising the PCR primers having thenucleotide sequence of SEQ ID No. 10 and SEQ ID No. 11.

The invention also relates to a kit for identifying elite event EE-GH5in biological samples, which kit comprises a specific probe having asequence which corresponds (or is complementary to) a sequence havingbetween 80% and 100% sequence identity with a specific region of EE-GH5.Preferably, the sequence of the probe corresponds to a specific regioncomprising part of the 5′ or 3′ flanking region of EE-GH5 or to a regioncorresponding to the rearrangement of foreign DNA specific for EE-GH5.Most preferably the specific probe has (or is complementary to) asequence having between 80% and 100% sequence identity to the sequencebetween nucleotide 78 to 119 of SEQ ID No 1 or SEQ ID No. 2 fromnucleotide 20 to 61 or SEQ ID 12 from nucleotide 52 to nucleotide 88.

According to another aspect of the invention, DNA sequences aredisclosed comprising the insertion site of the event and sufficientlength of polynucleotides of both the cotton genomic DNA and the foreignDNA (transgene), so as to be useful as primer or probe for the detectionof EE-GH5. Such sequences may comprise at least 9 nucleotides of thecotton genomic DNA and a similar number of nucleotides of the foreignDNA (transgene) of EE-GH5 therewith at each side of the insertion siterespectively. Most preferably, such DNA sequences comprise at least 9nucleotides of the cotton genomic DNA and a similar number ofnucleotides of the foreign DNA contiguous with the insertion site in SEQID NO: 1 or SEQ ID NO: 2.

The methods and kits encompassed by the present invention can be usedfor different purposes such as, but not limited to the following: toidentify the presence or absence of EE-GH5 in plants, plant material orin products such as, but not limited to food or feed products (fresh orprocessed) comprising or derived from plant material; additionally oralternatively, the methods and kits of the present invention can be usedto identify transgenic plant material for purposes of segregationbetween transgenic and non-transgenic material; additionally oralternatively, the methods and kits of the present invention can be usedto determine the quality (i.e. percentage pure material) of plantmaterial comprising EE-GH5.

The invention further relates to the 5′ and/or 3′ flanking regions ofEE-GH5 as well as to the specific primers and probes developed from the5′ and/or 3′ flanking sequences of EE-GH5. The invention also relates tothe EE-GH5 specific rearrangement region, as well as to specific primersor probes developed from that region.

The invention also relates to genomic DNA obtained from plantscomprising elite event EE-GH5. Such genomic DNA may be used as referencecontrol material in the identification assays herein described.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Examples, not intended to limit the invention to specificembodiments described, may be understood in conjunction with theaccompanying Figure, incorporated herein by reference, in which:

FIG. 1: Represents Schematically the Relationship Between the CitedNucleotide Sequences and Primers.

black bar: foreign DNA; striped black bar: rearrangement in the foreignDNA specific for EE-GH5; light bar: DNA of plant origin; striped lightbar: pre-insertion target deletion; arrows: oligonucleotide primers, thefigures under the bars represent nucleotide positions; (c) refers tocomplement of the indicated nucleotide sequence.

FIG. 2: Represents Results Obtained by the PCR Identification ProtocolDeveloped for EE-GH5.

Loading sequence of the gel: Lane1: Molecular weight marker (100 bpladder); lanes 2 to 3: DNA samples from cotton plants comprising thetransgenic event EE-GH5; lanes 4 to 10: DNA samples from transgeniccotton plants not comprising elite event EE-GH5, but comprising asimilar insect-resistance gene; lane 11: no template DNA control; lane12: molecular weight marker.

FIG. 3: Represents Results Obtained by the Zygosity Scoring PCR ProtocolDeveloped for EE-GH5.

Loading sequence of the gel: Lane1: Molecular weight marker (100 bpladder); lanes 2 and 3: DNA samples from cotton plants comprising thetransgenic event EE-GH5 in heterozygous form; lanes 4: Fail Lane 5:control DNA sample from wild-type cotton plant; lanes 6-7: DNA samplefrom cotton plants comprising the transgenic event EE-GH5 in homozygousform lane 8: no template DNA control; lane 9: molecular weight marker.

DETAILED DESCRIPTION

The incorporation of a recombinant DNA molecule in the plant genometypically results from transformation of a cell or tissue. Theparticular site of incorporation is usually due to “random” integration.

The DNA introduced into the plant genome as a result of transformationof a plant cell or tissue with a recombinant DNA or “transforming DNA”,and originating from such transforming DNA is hereinafter referred to as“foreign DNA” comprising one or more “transgenes”. “Plant DNA” in thecontext of the present invention will refer to DNA originating from theplant which is transformed. Plant DNA will usually be found in the samegenetic locus in the corresponding wild-type plant. The foreign DNA canbe characterized by the location and the configuration at the site ofincorporation of the recombinant DNA molecule in the plant genome. Thesite in the plant genome where a recombinant DNA has been inserted isalso referred to as the “insertion site” or “target site”. Insertion ofthe recombinant DNA into the region of the plant genome referred to as“pre-insertion plant DNA” can be associated with a deletion of plantDNA, referred to as “target site deletion”. A “flanking region” or“flanking sequence” as used herein refers to a sequence of at least 20bp, preferably at least 50 bp, and up to 5000 bp of DNA different fromthe introduced DNA, preferably DNA from the plant genome which islocated either immediately upstream of and contiguous with orimmediately downstream of and contiguous with the foreign DNA.Transformation procedures leading to random integration of the foreignDNA will result in transformants with different flanking regions, whichare characteristic and unique for each transformant. When therecombinant DNA is introduced into a plant through traditional crossing,its insertion site in the plant genome, or its flanking regions willgenerally not be changed. An “insertion region” as used herein refers tothe region corresponding to the region of at least 40 bp, preferably atleast 100 bp, and up to 10000 bp, encompassed by the sequence whichcomprises the upstream and/or the downstream flanking region of aforeign DNA in the plant genome. Taking into consideration minordifferences due to mutations within a species, an insertion region willretain, upon crossing into a plant of the same species, at least 85%,preferably 90%, more preferably 95%, and most preferably 100% sequenceidentity with the sequence comprising the upstream and downstreamflanking regions of the foreign DNA in the plant originally obtainedfrom transformation.

An event is defined as a (artificial) genetic locus that, as a result ofgenetic engineering, carries a transgene comprising at least one copy ofa gene of interest. The typical allelic states of an event are thepresence or absence of the foreign DNA. An event is characterizedphenotypically by the expression of the transgene. At the genetic level,an event is part of the genetic make-up of a plant. At the molecularlevel, an event can be characterized by the restriction map (e.g. asdetermined by Southern blotting), by the upstream and/or downstreamflanking sequences of the transgene, the location of molecular markersand/or the molecular configuration of the transgene. Usuallytransformation of a plant with a transforming DNA comprising at leastone gene of interest leads to a population of transformants comprising amultitude of separate events, each of which is unique.

An elite event, as used herein, is an event which is selected from agroup of events, obtained by transformation with the same transformingDNA or by back-crossing with plants obtained by such transformation,based on the expression and stability of the transgene(s) and itscompatibility with optimal agronomic characteristics of the plantcomprising it. Thus the criteria for elite event selection are one ormore, preferably two or more, advantageously all of the following:

a) That the presence of the foreign DNA does not compromise otherdesired characteristics of the plant, such as those relating toagronomic performance or commercial value;b) That the event is characterized by a well defined molecularconfiguration which is stably inherited and for which appropriate toolsfor identity control can be developed;c) That the gene(s) of interest show(s) a correct, appropriate andstable spatial and temporal phenotypic expression, both in heterozygous(or hemizygous) and homozygous condition of the event, at a commerciallyacceptable level in a range of environmental conditions in which theplants carrying the event are likely to be exposed in normal agronomicuse.

It is preferred that the foreign DNA is associated with a position inthe plant genome that allows easy introgression into desired commercialgenetic backgrounds.

The status of an event as an elite event is confirmed by introgressionof the elite event in different relevant genetic backgrounds andobserving compliance with one, two or all of the criteria e.g. a), b)and c) above.

An “elite event” thus refers to a genetic locus comprising a foreignDNA, which answers to the above-described criteria. A plant, plantmaterial or progeny such as seeds can comprise one or more elite eventsin its genome.

The tools developed to identify an elite event or the plant, plantmaterial comprising an elite event, or products which comprise plantmaterial comprising the elite event are based on the specific genomiccharacteristics of the elite event, such as, a specific restriction mapof the genomic region comprising the foreign DNA, molecular markers orthe sequence of the flanking region(s) of the foreign DNA.

Once one or both of the flanking regions of the foreign DNA have beensequenced, primers and probes can be developed which specificallyrecognize this (these) sequence(s) in the nucleic acid (DNA or RNA) of asample by way of a molecular biological technique. For instance a PCRmethod can be developed to identify the elite event in biologicalsamples (such as samples of plants, plant material or productscomprising plant material). Such a PCR is based on at least two specific“primers” one recognizing a sequence within the 5′ or 3′ flanking regionof the elite event and the other recognizing a sequence within theforeign DNA. The primers preferably have a sequence of between 15 and 35nucleotides which under optimized PCR conditions “specificallyrecognize” a sequence within the 5′ or 3′ flanking region of the eliteevent and the foreign DNA of the elite event respectively, so that aspecific fragment (“integration fragment” or discriminating amplicon) isamplified from a nucleic acid sample comprising the elite event. Thismeans that only the targeted integration fragment, and no other sequencein the plant genome or foreign DNA, is amplified under optimized PCRconditions.

PCR primers suitable for the invention may be the following:

-   -   oligonucleotides ranging in length from 17 nt to about 100 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides selected from        the 5′ flanking sequence (SEQ ID No 1 from nucleotide 1 to        nucleotide 98 or SEQ ID No 16 from nucleotide 1 to 563) at their        3′ end (primers recognizing 5′ flanking sequences); or    -   oligonucleotides ranging in length from 17 nt to about 200 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 consecutive nucleotides, selected        from the 3′ flanking sequence (complement of SEQ ID No 2 from        nucleotide 41 to nucleotide 452) at their 3′ end (primers        recognizing 3′ flanking sequences); or    -   oligonucleotides ranging in length from 17 nt to about 200 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 nucleotides selected from the        inserted DNA sequences (complement of SEQ ID No 1 from        nucleotide 99 to nucleotide 334) at their 3′ end (primers        recognizing foreign DNA) or    -   oligonucleotides ranging in length from 17 nt to about 40 nt,        comprising a nucleotide sequence of at least 17 consecutive        nucleotides, preferably 20 nucleotides selected from the        inserted DNA sequences (SEQ ID No 2 from nucleotide 1 to        nucleotide 40)

The primers may of course be longer than the mentioned 17 consecutivenucleotides, and may e.g. be 20, 21, 30, 35, 50, 75, 100, 150, 200 ntlong or even longer. The primers may entirely consist of nucleotidesequence selected from the mentioned nucleotide sequences of flankingsequences and foreign DNA sequences. However, the nucleotide sequence ofthe primers at their 5′ end (i.e. outside of the 3′-located 17consecutive nucleotides) is less critical. Thus, the 5′ sequence of theprimers may consist of a nucleotide sequence selected from the flankingsequences or foreign DNA, as appropriate, but may contain several (e.g.1, 2, 5, 10 mismatches). The 5′ sequence of the primers may evenentirely consist of a nucleotide sequence unrelated to the flankingsequences or foreign DNA, such as e.g. a nucleotide sequencerepresenting restriction enzyme recognition sites. Such unrelatedsequences or flanking DNA sequences with mismatches should preferably benot longer than 100, more preferably not longer than 50 or even 25nucleotides.

Moreover, suitable primers may comprise or consist of a nucleotidesequence at their 3′ end spanning the joining region between the plantDNA derived sequences and the foreign DNA sequences (located atnucleotides 98-99 in SEQ ID No 1 and nucleotides 40-41 in SEQ ID No 2)provided the mentioned 3′-located 17 consecutive nucleotides are notderived exclusively from either the foreign DNA or plant-derivedsequences in SEQ ID No 1 or 2.

It will also be immediately clear to the skilled artisan that properlyselected PCR primer pairs should also not comprise sequencescomplementary to each other.

For the purpose of the invention, the “complement of a nucleotidesequence represented in SEQ ID No: X” is the nucleotide sequence whichcan be derived from the represented nucleotide sequence by replacing thenucleotides through their complementary nucleotide according toChargaff's rules (A

T; G

C) and reading the sequence in the 5′ to 3′ direction, i.e in oppositedirection of the represented nucleotide sequence.

Examples of suitable primers are the oligonucleotide sequences of SEQ IDNo 7 or SEQ ID No 17 (5′ flanking sequence recognizing primers), SEQ IDNo 8, SEQ ID No 18 or SEQ ID No 19 (foreign DNA recognizing primers foruse with the 5′ flanking sequence recognizing primers), SEQ ID No 3(foreign DNA recognizing primers for use with the 3′ flanking sequencerecognizing primers), SEQ ID No 4, SEQ ID No 5, SEQ ID No 6 (3′ flankingsequence recognizing primers) and SEQ ID Nos 10 and 11 (primersrecognizing the specific foreign DNA rearrangement in EE-GH5).

Other examples of suitable oligonucleotide primers comprise at their 3′end the following sequences or consist of such sequences:

a. 5′ flanking sequence recognizing primers:

-   -   the nucleotide sequence of SEQ ID No 1 from nucleotide 58 to        nucleotide 77    -   the nucleotide sequence of SEQ ID No 16 from nucleotide 85 to        nucleotide 104    -   the nucleotide sequence of SEQ ID No 16 from nucleotide 150 to        nucleotide 166    -   the nucleotide sequence of SEQ ID No 16 from nucleotide 150 to        nucleotide 171    -   the nucleotide sequence of SEQ ID No 16 from nucleotide 154 to        nucleotide 171    -   the nucleotide sequence of SEQ ID No 16 from nucleotide 87 to        nucleotide 104    -   the nucleotide sequence of SEQ ID No 16 from nucleotide 189 to        nucleotide 206        b. foreign DNA sequence recognizing primers for use with 5′        flanking sequence recognizing primers:    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 149 to nucleotide 170    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 169 to nucleotide 186    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 193 to nucleotide 212    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 161 to nucleotide 178    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 194 to nucleotide 211    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 193 to nucleotide 211    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 163 to nucleotide 185    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 194 to nucleotide 210    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 193 to nucleotide 210    -   the complement of the nucleotide sequence of SEQ ID No 1 from        nucleotide 193 to nucleotide 209        c. 3′ flanking sequence recognizing primers:    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 149 to nucleotide 168    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 154 to nucleotide 173    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 140 to nucleotide 159    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 141 to nucleotide 160    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 147 to nucleotide 166    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 148 to nucleotide 167    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 149 to nucleotide 167    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 153 to nucleotide 172    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 154 to nucleotide 174    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 160 to nucleotide 177    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 232 to nucleotide 251    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 140 to nucleotide 160    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 141 to nucleotide 161    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 147 to nucleotide 165    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 149 to nucleotide 166    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 148 to nucleotide 166    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 147 to nucleotide 167    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 148 to nucleotide 168    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 153 to nucleotide 173    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 154 to nucleotide 175    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 156 to nucleotide 175    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 232 to nucleotide 250    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 140 to nucleotide 157    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 140 to nucleotide 161    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 141 to nucleotide 162    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 148 to nucleotide 165    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 149 to nucleotide 165    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 147 to nucleotide 168    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 148 to nucleotide 169    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 156 to nucleotide 174    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 153 to nucleotide 174    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 232 to nucleotide 249    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 234 to nucleotide 251    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 141 to nucleotide 163    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 148 to nucleotide 164    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 147 to nucleotide 169    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 153 to nucleotide 175    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 156 to nucleotide 177    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 181 to nucleotide 198    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 181 to nucleotide 202    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 234 to nucleotide 250    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 140 to nucleotide 162    -   the complement of the nucleotide sequence of SEQ ID No 2 from        nucleotide 181 to nucleotide 203        d. foreign DNA sequence recognizing primers for use with 3′        flanking sequence recognizing primers:    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 30 to        nucleotide 49    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 30 to        nucleotide 48    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 32 to        nucleotide 48    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 32 to        nucleotide 49    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 31 to        nucleotide 49    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 31 to        nucleotide 48    -   the nucleotide sequence of SEQ ID No 2 from nucleotide 30 to        nucleotide 46

As used herein, “the nucleotide sequence of SEQ ID No. Z from position Xto position Y” indicates the nucleotide sequence including bothnucleotide endpoints.

Preferably, the amplified fragment has a length of between 50 and 500nucleotides, most preferably of between 100 and 350 nucleotides. Thespecific primers may have a sequence which is between 80 and 100%identical to a sequence within the 5′ or 3′ flanking region of the eliteevent and the foreign DNA of the elite event, respectively, provided themismatches still allow specific identification of the elite event withthese primers under optimized PCR conditions. The range of allowablemismatches however, can easily be determined experimentally and areknown to a person skilled in the art.

The following table exemplifies the sizes of expected DNA amplicons (orintegration fragments) with selected pairs of PCR primers.

Length Primer 1 From position Primer 2 To position amplicon HVH024 58DPA312 186 128 GHI057 1 MAE115 175 175 GHI057 1 HVH022 251 251 GHI057 1GHI065 369 369

Detection of integration fragments can occur in various ways e.g. viasize estimation after gel analysis. The integration fragments may alsobe directly sequenced. Other sequence specific methods for detection ofamplified DNA fragments are also known in the art.

As the sequence of the primers and their relative location in the genomeare unique for the elite event, amplification of the integrationfragment will occur only in biological samples comprising (the nucleicacid of) the elite event. Preferably when performing a PCR to identifythe presence of EE-GH5 in unknown samples, a control is included of aset of primers with which a fragment within a “housekeeping gene” of theplant species of the event can be amplified. Housekeeping genes aregenes that are expressed in most cell types and which are concerned withbasic metabolic activities common to all cells. Preferably, the fragmentamplified from the housekeeping gene is a fragment which is larger thanthe amplified integration fragment. Depending on the samples to beanalyzed, other controls can be included.

Standard PCR protocols are described in the art, such as in ‘PCRApplications Manual” (Roche Molecular Biochemicals, 2nd Edition, 1999)and other references. The optimal conditions for the PCR, including thesequence of the specific primers, is specified in a “PCR identificationprotocol” for each elite event. It is however understood that a numberof parameters in the PCR identification protocol may need to be adjustedto specific laboratory conditions, and may be modified slightly toobtain similar results. For instance, use of a different method forpreparation of DNA may require adjustment of, for instance, the amountof primers, polymerase and annealing conditions used. Similarly, theselection of other primers may dictate other optimal conditions for thePCR identification protocol. These adjustments will however be apparentto a person skilled in the art, and are furthermore detailed in currentPCR application manuals such as the one cited above.

Alternatively, specific primers can be used to amplify an integrationfragment that can be used as a “specific probe” for identifying EE-GH5in biological samples. Contacting nucleic acid of a biological sample,with the probe, under conditions which allow hybridization of the probewith its corresponding fragment in the nucleic acid, results in theformation of a nucleic acid/probe hybrid. The formation of this hybridcan be detected (e.g. labeling of the nucleic acid or probe), wherebythe formation of this hybrid indicates the presence of EE-GH5. Suchidentification methods based on hybridization with a specific probe(either on a solid phase carrier or in solution) have been described inthe art. The specific probe is preferably a sequence which, underoptimized conditions, hybridizes specifically to a region within the 5′or 3′ flanking region of the elite event and preferably also comprisingpart of the foreign DNA contiguous therewith (hereinafter referred to as“specific region”). Preferably, the specific probe comprises a sequenceof between 50 and 500 bp, preferably of 100 to 350 bp which is at least80%, preferably between 80 and 85%, more preferably between 85 and 90%,especially preferably between 90 and 95%, most preferably between 95%and 100% identical (or complementary) to the nucleotide sequence of aspecific region. Preferably, the specific probe will comprise a sequenceof about 15 to about 100 contiguous nucleotides identical (orcomplementary) to a specific region of the elite event.

Oligonucleotides suitable as PCR primers for detection of the eliteevent EE-GH5 can also be used to develop a PCR-based protocol todetermine the zygosity status of the elite event. To this end, twoprimers recognizing the wild-type locus are designed in such a way thatthey are directed towards each other and have the insertion site locatedin between the primers. These primers may be primers specificallyrecognizing the 5′ and 3′ flanking sequences contained within SEQ ID NO1 or 2, respectively. These primers may also be primers specificallyrecognizing the 5′ or 3′ flanking sequence (such as a primer having thenucleotide sequence of SEQ ID No 7 or SEQ ID Nos 4-6) as well as aprimer recognizing the region deleted during EE-GH5 insertion within thepre-insertion plant DNA). This set of primers, together with a thirdprimer complementary to transforming DNA sequences (such as a primerhaving the nucleotide sequence of SEQ ID No 3) allow diagnostic PCRamplification of the EE-GH5 specific locus, as well as of the wt locus.If the plant is homozygous for the transgenic locus or the correspondingwt locus, the diagnostic PCR will give rise to a single PCR producttypical, preferably typical in length, for either the transgenic or wtlocus. If the plant is hemizygous for the transgenic locus, two locusspecific PCR products will appear, reflecting both the amplification ofthe transgenic and wt locus.

Furthermore, detection methods specific for elite event EE-GH5 whichdiffer from PCR based amplification methods can also be developed usingthe elite event specific sequence information provided herein. Suchalternative detection methods include linear signal amplificationdetection methods based on invasive cleavage of particular nucleic acidstructures, also known as Invader™ technology, (as described e.g. inU.S. Pat. Nos. 5,985,557 “Invasive Cleavage of Nucleic Acids”, 6,001,567“Detection of Nucleic Acid sequences by Invader Directed Cleavage,incorporated herein by reference). To this end, the target sequence mayhybridized with a labeled first nucleic acid oligonucleotide comprisingthe nucleotide sequence of SEQ ID No 1 from nucleotide 99 to nucleotide116 or its complement or said labeled nucleic acid probe comprising thenucleotide sequence of SEQ ID No 2 from nucleotide 23 to nucleotide 40or its complement and is further hybridized with a second nucleic acidoligonucleotide comprising the nucleotide sequence of SEQ ID No 1 fromnucleotide 81 to nucleotide 98 or its complement or said labeled nucleicacid probe comprising the nucleotide sequence of SEQ ID No 2 fromnucleotide 41 to nucleotide 58 or its complement, wherein the first andsecond oligonucleotide overlap by at least one nucleotide. The duplex ortriplex structure which is produced by this hybridization allowsselective probe cleavage with an enzyme (Cleavase®) leaving the targetsequence intact. The cleaved labeled probe is subsequently detected,potentially via an intermediate step resulting in further signalamplification.

A “kit” as used herein refers to a set of reagents for the purpose ofperforming the method of the invention, more particularly, theidentification of the elite event EE-GH5 in biological samples or thedetermination of the zygosity status of EE-GH5 containing plantmaterial. More particularly, a preferred embodiment of the kit of theinvention comprises at least one or two specific primers, as describedabove for identification of the elite event, or three specific primersfor the determination of the zygosity status. Optionally, the kit canfurther comprise any other reagent described herein in the PCRidentification protocol. Alternatively, according to another embodimentof this invention, the kit can comprise a specific probe, as describedabove, which specifically hybridizes with nucleic acid of biologicalsamples to identify the presence of EE-GH5 therein. Optionally, the kitcan further comprise any other reagent (such as but not limited tohybridizing buffer, label) for identification of EE-GH5 in biologicalsamples, using the specific probe.

The kit of the invention can be used, and its components can bespecifically adjusted, for purposes of quality control (e.g., purity ofseed lots), detection of the presence or absence of the elite event inplant material or material comprising or derived from plant material,such as but not limited to food or feed products.

As used herein, “sequence identity” with regard to nucleotide sequences(DNA or RNA), refers to the number of positions with identicalnucleotides divided by the number of nucleotides in the shorter of thetwo sequences. The alignment of the two nucleotide sequences isperformed by the Wilbur and Lipmann algorithm (Wilbur and Lipmann, 1983,Proc. Nat. Acad. Sci. USA 80:726) using a window-size of 20 nucleotides,a word length of 4 nucleotides, and a gap penalty of 4.Computer-assisted analysis and interpretation of sequence data,including sequence alignment as described above, can, e.g., beconveniently performed using the sequence analysis software package ofthe Genetics Computer Group (GCG, University of Wisconsin Biotechnologycenter). Sequences are indicated as “essentially similar” when suchsequences have a sequence identity of at least about 75%, particularlyat least about 80%, more particularly at least about 85%, quiteparticularly about 90%, especially about 95%, more especially about100%. It is clear than when RNA sequences are said to be essentiallysimilar or have a certain degree of sequence identity with DNAsequences, thymidine (T) in the DNA sequence is considered equal touracil. (U) in the RNA sequence.

The term “primer” as used herein encompasses any nucleic acid that iscapable of priming the synthesis of a nascent nucleic acid in atemplate-dependent process, such as PCR. Typically, primers areoligonucleotides from 10 to 30 nucleotides, but longer sequences can beemployed. Primers may be provided in double-stranded form, though thesingle-stranded form is preferred. Probes can be used as primers, butare designed to bind to the target DNA or RNA and need not be used in anamplification process.

The term “recognizing” as used herein when referring to specificprimers, refers to the fact that the specific primers specificallyhybridize to a nucleic acid sequence in the elite event under theconditions set forth in the method (such as the conditions of the PCRidentification protocol), whereby the specificity is determined by thepresence of positive and negative controls.

The term “hybridizing” as used herein when referring to specific probes,refer to the fact that the probe binds to a specific region in thenucleic acid sequence of the elite event under standard stringencyconditions. Standard stringency conditions as used herein refers to theconditions for hybridization described herein or to the conventionalhybridizing conditions as described by Sambrook et al., 1989 (MolecularCloning: A Laboratory Manual, Second Edition, Cold Spring HarbourLaboratory Press, NY) which for instance can comprise the followingsteps: 1) immobilizing plant genomic DNA fragments on a filter, 2)prehybridizing the filter for 1 to 2 hours at 42° C. in 50% formamide,5×SSPE, 2×Denhardt's reagent and 0.1% SDS, or for 1 to 2 hours at 68° C.in 6×SSC, 2×Denhardt's reagent and 0.1% SDS, 3) adding the hybridizationprobe which has been labeled, 4) incubating for 16 to 24 hours, 5)washing the filter for 20 min. at room temperature in 1×SSC, 0.1% SDS,6) washing the filter three times for 20 min. each at 68° C. in 0.2×SSC,0.1% SDS, and 7) exposing the filter for 24 to 48 hours to X-ray film at−70° C. with an intensifying screen.

As used in herein, a biological samples is a sample of a plant, plantmaterial or products comprising plant material. The term “plant” isintended to encompass cotton (Gossypium hirsitum) plant tissues, at anystage of maturity, as well as any cells, tissues, or organs taken fromor derived from any such plant, including without limitation, any seeds,leaves, stems, flowers, roots, single cells, gametes, cell cultures,tissue cultures or protoplasts. “Plant material”, as used herein refersto material which is obtained or derived from a plant. Productscomprising plant material relate to food, feed or other products whichare produced using plant material or can be contaminated by plantmaterial. It is understood that, in the context of the presentinvention, such biological samples are tested for the presence ofnucleic acids specific for EE-GH5, implying the presence of nucleicacids in the samples. Thus the methods referred to herein foridentifying elite event EE-GH5 in biological samples, relate to theidentification in biological samples of nucleic acids which comprise theelite event.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps, reagents or componentsas referred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e.g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i.e., be embedded in a larger nucleic acid orprotein. A chimeric gene comprising a DNA sequence which is functionallyor structurally defined, may comprise additional DNA sequences, etc.

The present invention also relates to the development of an elite eventEE-GH5 in cotton to the plants comprising this event, the progenyobtained from these plants and to the plant cells, or plant materialderived from this event. Plants comprising elite event EE-GH5 wereobtained described in example 1.

Cotton plants or plant material comprising EE-GH5 can be identifiedaccording to the PCR identification protocol described for EE-GH5 inExample 2. Briefly, cotton genomic DNA present in the biological sampleis amplified by PCR using a primer which specifically recognizes asequence within the 5′ or 3′ flanking sequence of EE-GH5 such as theprimer with the sequence of SEQ ID NO: 6, and a primer which recognizesa sequence in the foreign DNA, such as the primer with the sequence ofSEQ ID NO: 3. DNA primers which amplify part of an endogenous cottonsequence are used as positive control for the PCR amplification. If uponPCR amplification, the material yields a fragment of the expected size,the material contains plant material from a cotton plant harboring eliteevent EE-GH5.

Plants harboring EE-GH5 are characterized by their insect resistance, aswell as by their glufosinate tolerance. Plants harboring EE-GH5 are alsocharacterized by having agronomical characteristics that are comparableto commercially available varieties of cotton in the US, in the absenceof insect pressure. It has been observed that the presence of a foreignDNA in the insertion region of the cotton plant genome described herein,confers particularly interesting phenotypic and molecularcharacteristics to the plants comprising this event.

The following examples describe the identification of elite event EE-GH5and the development of tools for the specific identification of eliteevent EE-GH5 in biological samples.

Unless states otherwise in the Examples, all recombinant techniques arecarried out according to standard protocols as described in “Sambrook Jand Russell D W (eds.) (2001) Molecular Cloning: A Laboratory Manual,3rd Edition, Cold Spring Harbor Laboratory Press, New York” and in“Ausubel F A, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J Aand Struhl K (eds.) (2006) Current Protocols in Molecular Biology. JohnWiley & Sons, New York”.

Standard materials and references are described in “Croy R D D (ed.)(1993) Plant Molecular Biology LabFax, BIOS Scientific Publishers Ltd.,Oxford and Blackwell Scientific Publications, Oxford” and in “Brown T A,(1998) Molecular Biology LabFax, 2nd Edition, Academic Press, SanDiego”. Standard materials and methods for polymerase chain reactions(PCR) can be found in “McPherson M J and Møller S G (2000) PCR (TheBasics), BIOS Scientific Publishers Ltd., Oxford” and in “PCRApplications Manual, 3rd Edition (2006), Roche Diagnostics GmbH,Mannheim or www.roche-applied-science.com”

In the description and examples, reference is made to the followingsequences:

-   SEQ ID No. 1: nucleotide sequence comprising a 5′ flanking region of    EE-GH5-   SEQ ID No. 2: nucleotide sequence comprising a 3′ flanking region of    EE-GH5-   SEQ ID No. 3: primer GHI057-   SEQ ID No. 4: primer MAE115-   SEQ ID No. 5: primer HVH022-   SEQ ID No. 6: primer GHI065-   SEQ ID No. 7: primer HVH024-   SEQ ID No. 8: primer DPA312-   SEQ ID No. 9: primer GHI066-   SEQ ID No. 10: primer GHI047-   SEQ ID No. 11: primer GHI048-   SEQ ID No. 12: Nucleotide sequence of the fragment amplified from    cotton DNA comprising EE-GH5 using GHI047 and GHI048-   SEQ ID No. 13: primer 1 for amplification of control fragment-   SEQ ID No. 14: primer 2 for amplification of control fragment-   SEQ ID No. 15: Nucleotide sequence of the plant genomic target    sequence prior to insertion of EE-GH5-   SEQ ID No. 16: Nucleotide sequence comprising a 5′ flanking region    of EE-GH5 (long)-   SEQ ID No. 17: primer HVH036-   SEQ ID No. 18: primer SHA028-   SEQ ID No. 19: primer MAE067

EXAMPLES 1. Identification of Elite Event EE-GH5

Insect-resistant cotton was developed by transformation of cotton with avector comprising the coding sequence of a modified cry1ab gene underthe control of a Subterranean Clover stunt virus S7 promoter.

Elite event EE-GH5 was selected based on an extensive selectionprocedure based on good expression and stability of the insectresistance gene and its compatibility with optimal agronomiccharacteristics such as plant height, height to node, boll retention,stand, vigor, fiber length, fiber strength and lint yield wereevaluated.

The selected event was introduced into different commercial geneticbackgrounds, and results of field trials on different locations werecompared. Plants were challenged with insect pests using differenttreatments. Plants exhibited good insect control.

Furthermore, the event had normal leaf, flower and boll morphology,excellent fertility, and showed no disease or abnormal insectsusceptibility in multiple genetic backgrounds. During introgressioninto multiple genetic backgrounds no aberrant problems or abnormalitieswere observed.

2. Identification of the Flanking Regions of Elite Event EE-GH5

The sequence of the regions flanking the foreign DNA in the EE-GH5 eliteevent was determined using the thermal asymmetric interlaced (TAIL-) PCRmethod described by Liu et al. (1995, Plant J. 8(3):457-463) wherepossible. This method utilizes three nested primers in successivereactions together with a shorter arbitrary degenerate primer so thatthe relative amplification efficiencies of specific and non-specificproducts can be thermally controlled. The specific primers were selectedfor annealing to the border of the foreign DNA and based on theirannealing conditions. A small amount (5 μl) of unpurified, secondary andtertiary, PCR products were analyzed on a 1% agarose gel. The tertiaryPCR product was purified and sequenced.

2.1. Right (5′) Flanking Region

The fragment identified as comprising the 5′ flanking region wassequenced (SEQ ID No. 1). The sequence between nucleotide 1 and 98corresponds to plant DNA, while the sequence between nucleotide 99 and334 corresponds to foreign DNA. A longer fragment comprising the 5′flanking region was also sequenced (SEQ ID No. 16). The sequence betweennucleotide 1 and 563 corresponds to plant DNA, while the sequencebetween nucleotide 564 and 799 corresponds to foreign DNA

2.2. Left (3′) Flanking Region

The fragment identified as comprising the 3′ flanking region obtained bythe TAIL-PCR method was sequenced (SEQ ID No. 2). The sequence betweennucleotide 1 and 40 corresponds to foreign DNA, while the sequencebetween nucleotide 41 and 452 corresponds to plant DNA.

2.3. Region Comprising EE-GH5 Specific Rearrangement in Foreign DNA.

The nucleotide sequence of a region comprising the EE-GH5 specificrearrangement in the foreign DNA was identified (SEQ ID No. 12). Theregion between nucleotide positions 52 and 88 were identified as beingrearranged when compared with the foreign DNA used to obtaintransformants from which elite event EE-GH5 was selected.

3. Development of Polymerase Chain Reaction Identification Protocols forEE-GH5 3.1. Primers

Specific primers were developed which recognize sequences within theelite event.

More particularly, two primers were developed which recognize sequencesflanking the EE-GH5 specific rearrangement in the foreign DNA. Theprimers span a sequence of about 262 bp. The following primers werefound to give particularly clear and reproducible results in a PCRreaction on EE-GH5 DNA:

(SEQ ID No.: 10) GHI047: 5′-TgC.CTC.TTg.AAC.TgT.AgC-3′ (SEQ ID No.: 11)GHI048: 5′-ACT.TgC.AgT.TgC.TgA.TgA.Tg-3′

Alternatively, a primer was developed which recognizes a sequence withinthe 5′ flanking region of EE-GH5. A second primer was then selectedwithin the sequence of the foreign DNA so that the primers span asequence of about 369 nucleotides. The following primers were found togive particularly clear and reproducible results in a PCR reaction onEE-GH5 DNA:

(SEQ ID No.: 6) GHI065: 5′-ggg.CCg.gAT.AAA.ATT.AgC.CT-3′(target: plant DNA) (SEQ ID No.: 3) GHI057:5′-ATA.gCg.CgC.AAA.CTA.ggA-3′ (target: insert DNA)

Primers targeting an endogenous sequence are preferably included in thePCR cocktail. These primers serve as an internal control in unknownsamples and in the DNA positive control. A positive result with theendogenous primer-pair demonstrates that there is ample DNA of adequatequality in the genomic DNA preparation for a PCR product to begenerated. The endogenous primers were selected to recognize ahousekeeping gene in cotton:

(SEQ ID No.: 13) GHI001: 5′-AAC.CTA.ggC.TgC.TgA.Agg.AgC-3′(SEQ ID No.: 14) GHI002: 5′-CAA.CTC.CTC.CAg.TCA.TCT.CCg-3′

3.2. Amplified Fragments

The expected amplified fragments in the PCR reaction are:

For primer pair GHI001-GHI002: 445 bp (endogenous control)For primer pair GH1047-GHI048: 262 bp (EE-GH5 elite Event)For primer pair GHI057-GH1065: 369 bp (EE-GH5 elite Event)

3.3. Template DNA

Template DNA was prepared from a leaf punch according to Edwards et al.(Nucleic Acid Research, 19, p 1349, 1991). When using DNA prepared withother methods, a test run utilizing different amounts of template shouldbe done. Usually 50 ng of genomic template DNA yields the best results.

3.4. Assigned Positive and Negative Controls

To avoid false positives or negatives, it was determined that thefollowing positive and negative controls should be included in a PCRrun:

-   -   Master Mix control (DNA negative control). This is a PCR in        which no DNA is added to the reaction. When the expected result,        no PCR products, is observed this indicates that the PCR        cocktail was not contaminated with target DNA.    -   A DNA positive control (genomic DNA sample known to contain the        transgenic sequences). Successful amplification of this positive        control demonstrates that the PCR was run under conditions which        allow for the amplification of target sequences.    -   A wild-type DNA control. This is a PCR in which the template DNA        provided is genomic DNA prepared from a non-transgenic plant.        When the expected result, no amplification of a transgene PCR        product but amplification of the endogenous PCR product, is        observed this indicates that there is no detectable transgene        background amplification in a genomic DNA sample.

3.5. PCR Conditions

Optimal results were obtained under the following conditions:

-   -   the PCR mix for 25 μl reactions contains:        -   2.5 μl template DNA        -   2.5 μl 10× Amplification Buffer (supplied by the            manufacturer with the Taq polymerase)        -   0.5 μl 10 mM dNTP's        -   0.4 μl GHI001 (10 pmoles/μl)        -   0.4 μl GHI002 (10 pmoles/μl)        -   0.7 μl GHI047 (10 pmoles/μl) or GHI057 (PCR protocol 2)        -   0.7 μl GHI048 (10 pmoles/μl) or GHI065 (PCR protocol 2)        -   0.1 μl Taq DNA polymerase (5 units/μl)        -   water up to 25 μl    -   the thermocycling profile to be followed for optimal results is        the following:        -   4 min. at 95° C.    -   Followed by:        -   1 min. at 95° C.        -   1 min. at 57° C.        -   2 min. at 72° C.        -   For 5 cycles    -   Followed by:        -   30 sec. at 92° C.        -   30 sec. at 57° C.        -   1 min. at 72° C.        -   For 25 cycles    -   Followed by:        -   10 minutes at 72° C.

3.6. Agarose Gel Analysis

To optimally visualise the results of the PCR it was determined thatbetween 10 and 20 μl of the PCR samples should be applied on a 1.5%agarose gel (Tris-borate buffer) with an appropriate molecular weightmarker (e.g. 100 bp ladder PHARMACIA).

3.7. Validation of the Results

It was determined that data from transgenic plant DNA samples within asingle PCR run and a single PCR cocktail should not be acceptableunless 1) the DNA positive control shows the expected PCR products(transgenic and endogenous fragments), 2) the DNA negative control isnegative for PCR amplification (no fragments) and 3) the wild-type DNAcontrol shows the expected result (endogenous fragment amplification).

When following the PCR Identification Protocol for EE-GH5 as describedabove, lanes showing visible amounts of the transgenic and endogenousPCR products of the expected sizes, indicate that the correspondingplant from which the genomic template DNA was prepared, has inheritedthe EE-GH5 elite event. Lanes not showing visible amounts of either ofthe transgenic PCR products and showing visible amounts of theendogenous PCR product, indicate that the corresponding plant from whichthe genomic template DNA was prepared, does not comprise the eliteevent. Lanes not showing visible amounts of the endogenous andtransgenic PCR products, indicate that the quality and/or quantity ofthe genomic DNA didn't allow for a PCR product to be generated. Theseplants cannot be scored. The genomic DNA preparation should be repeatedand a new PCR run, with the appropriate controls, has to be performed.

3.8. Use of Discriminating PCR Protocol to Identify EE-GH5

Before attempting to screen unknowns, a test run, with all appropriatecontrols, has to be performed. The developed protocol might requireoptimization for components that may differ between labs (template DNApreparation, Taq DNA polymerase, quality of the primers, dNTP's,thermocyler, etc.).

Amplification of the endogenous sequence plays a key role in theprotocol. One has to attain PCR and thermocycling conditions thatamplify equimolar quantities of both the endogenous and transgenicsequence in a known transgenic genomic DNA template. Whenever thetargeted endogenous fragment is not amplified or whenever the targetedsequences are not amplified with the same ethidium bromide stainingintensities, as judged by agarose gel electrophoresis, optimization ofthe PCR conditions may be required.

Leaf material from a number of cotton plants, some of which comprisingEE-GH5 were tested according to the above-described protocol. Samplesfrom elite event EE-GH5 and from cotton wild-type were taken as positiveand negative controls, respectively.

FIG. 2 illustrates the result obtained with the elite event PCRidentification protocol 1 for EE-GH5 on a number of cotton plantsamples. The samples in lanes 2 to 3 were found to contain the eliteevent as the 305 bp band is detected, while the samples in lanes 4 to 10do not comprise EE-GH5. Lanes 4 and 10 comprise other cotton eliteevents (including plants comprising different insect tolerance chimericgenes); lane 11 represents the negative control (water) sample, andlanes 1 and 12 represent the Molecular Weight Marker (100 bp ladder).

4. Use of a Specific Integration Fragment as a Probe for Detection ofMaterial Comprising EE-GH5

A specific integration fragment of EE-GH5 is obtained by PCRamplification using specific primers GHI047 (SEQ ID No. 10) and GHI048(SEQ ID No. 11) or by chemical synthesis and is labeled. Thisintegration fragment is used as a specific probe for the detection ofEE-GH5 in biological samples. Nucleic acid is extracted from the samplesaccording to standard procedures. This nucleic acid is then contactedwith the specific probe under hybridization conditions which areoptimized to allow formation of a hybrid. The formation of the hybrid isthen detected to indicate the presence of EE-GH5 nucleic acid in thesample. Optionally, the nucleic acid in the samples is amplified usingthe specific primers prior to contact with the specific probe.Alternatively, the nucleic acid is labeled prior to contact with thespecific probe instead of the integration fragment. Optionally, thespecific probe is attached to a solid carrier (such as, but not limitedto a filter, strip or beads), prior to contact with the samples.

5. Protocol for the PCR-Based Determination of the Zygosity Status ofEE-GH5 Cotton Plant Material 5.1. Primers

Two primers recognizing the nucleotide sequences of the wild-type locusprior to insertion of the elite event, were designed in such a way thatthey are directed towards each other and have the insertion sitein-between. This set of primers, together with a third primercomplementary to foreign DNA sequences and directed towards the flankingDNA, allow simultaneous PCR amplification of the EE-GH5 locus as well asof the wt locus.

The following primers were found to give particularly clear andreproducible results in a zygosity scoring PCR reaction on EE-GH5 DNA:

(SEQ ID No.: 9) GHI066 5′-AgA.TAA.AAT.CgT.CAg.TgC.Tg-3′(target: plant DNA upstream of 5′ flanking sequence) (SEQ ID No.: 3)GHI057 5′-ATA.gCg.CgC.AAA.CTA.ggA-3′ (target: insert DNA)(SEQ ID No.: 6) GHI065 5′-ggg.CCg.gAT.AAA.ATT.AgC.CT-3′(target: plant DNA of the 3′ flanking sequence)

5.2. Amplified Fragments

The expected amplified fragments in the PCR reaction are:

For primer pair GHI065-GHI066: 517 bp (wild-type locus)For primer pair GH1057-GHI065: 369 bp (EE-GH5 locus)

5.3. Template DNA

Template DNA was prepared from a leaf punch according to Edwards et al.(Nucleic Acid Research, 19, p 1349, 1991). When using DNA prepared withother methods, a test run utilizing different amounts of template shouldbe done. Usually 50 ng of genomic template DNA yields the best results.

5.4. Assigned Positive and Negative Controls

To avoid false positives or negatives, it is advisable that thefollowing positive and negative controls should be included in a PCRrun:

-   -   Master Mix control (DNA negative control). This is a PCR in        which no DNA is added to the reaction. When the expected result,        no PCR products, is observed this indicates that the PCR        cocktail was not contaminated with target DNA.    -   A DNA positive control (genomic DNA sample known to contain the        transgenic sequences). Successful amplification of this positive        control demonstrates that the PCR was run under conditions which        allow for the amplification of target sequences.    -   A wild-type DNA control. This is a PCR in which the template DNA        provided is genomic DNA prepared from a non-transgenic plant.        When the expected result, no amplification of a transgene PCR        product but amplification of the endogenous PCR product, is        observed this indicates that there is no detectable transgene        background amplification in a genomic DNA sample.

5.5. PCR Conditions

Optimal results were obtained under the following conditions:

-   -   the PCR mix for 25 μl reactions contains:        -   x μl template DNA (150 ng)        -   2.5 μl 10× Amplification Buffer (supplied by the            manufacturer with the Taq polymerase)        -   0.5 μl 10 mM dNTP's        -   1.5 μl GHI053 (10 pmoles/μl)        -   1.0 μl GHI054 (10 pmoles/μl)        -   0.5 μl GHI041 (10 pmoles/μl)        -   0.1 μl Taq DNA polymerase (5 units/μl)        -   water up to 25 μl    -   the thermocycling profile to be followed for optimal results is        the following:        -   4 min. at 95° C.    -   Followed by:        -   1 min. at 95° C.        -   1 min. at 57° C.        -   2 min. at 72° C.        -   For 5 cycles    -   Followed by:        -   30 sec. at 92° C.        -   30 sec. at 57° C.        -   1 min. at 72° C.        -   For 25 cycles    -   Followed by:        -   10 minutes at 72° C.

5.6. Agarose Gel Analysis

To optimally visualise the results of the PCR it was determined thatbetween 10 and 20 μl of the PCR samples should be applied on a 1.5%agarose gel (Tris-borate buffer) with an appropriate molecular weightmarker (e.g. 100 bp ladder PHARMACIA).

5.7. Validation of the Results

Data from transgenic plant DNA samples within a single PCR run and asingle PCR Master Mix will not be acceptable unless:

-   -   the positive control shows the expected PCR products (transgenic        target amplification)    -   the wild-type-positive DNA control shows the expected result        (wild-type target amplification).    -   the negative control is negative for PCR amplification (no        fragments).

Lanes showing visible amounts of the transgenic PCR product of theexpected size and not showing visible amounts of the wild type PCRproduct, indicate that the corresponding plant from which the genomicDNA template was prepared, is homozygous for the transgenic genecassette.

Lanes showing visible amounts of the transgenic and wild type PCRproducts of the expected sizes, indicate that the corresponding plantfrom which the genomic template DNA was prepared, is hemizygous for thetransgenic gene cassette. Lanes not showing visible amounts of thetransgenic PCR product and showing visible amounts of the wild type PCRproduct, indicate that the corresponding plant from which the genomictemplate DNA was prepared, has not inherited the transgenic sequenceassayed for and is thus homozygous for the wild type locus.

Lanes not showing visible amounts of transgenic and wild type PCRproducts, indicate that the quality and/or quantity of the genomic DNAdidn't allow for a PCR product to be generated. These plants cannot bescored. The genomic DNA preparation should be repeated and a new PCRrun, with the appropriate controls, has to be performed.

5.8. Use of the Zygosity Scoring Protocol for Identification of ZygosityStatus in EE-GH5 containing plants.

FIG. 3 illustrates the result obtained with the zygosity scoring PCR forEE-GH5 on a number of cotton plant samples. The samples in lanes 2-3 and6-7 were found to contain the PCR fragment (369 bp) characteristic forelite event EE-GH5, while the samples in lanes 2, 3 and 5 contained thefragment characteristic for the presence of the wt locus. Lanes 6 and 7therefore contain EE-GH5 in homozygous form, lanes 2 and 3 containEE-GH5 in hemizygous form and lane 5 contains the wt locus in homozygousform (azygous for EE-GH5). Lane 8 represents the negative control(water) sample, and lanes 1 and 9 represent the Molecular Weight Marker(100 bp ladder).

6. Introgression of EE-GH5 into Preferred Cultivars

Elite event EE-GH5 is introduced by repeated back-crossing intocommercial cotton cultivars such as but not limited to FM5013, FM5015,FM5017, FM989, FM832, FM966 and FM958, FM989, FM958, FM832, FM991,FM819, FM800, FM960, FM966, FM981, FM5035, FM5044, FM5045, FM5013,FM5015, FM5017 or FM5024.

It is observed that the introgression of the elite event into thesecultivars does not significantly influence any of the desirablephenotypic or agronomic characteristics of these cultivars (no linkagedrag) while expression of the transgene, as determined by glufosinatetolerance, meets commercially acceptable levels. This confirms thestatus of event EE-GH5 as an elite event.

Elite event may be advantageously combined with other elite eventsavailable in the market, particularly other elite event insectresistance gene for the purpose of insect resistance management, such asbut not limited to event 3006-210-23; (USDA aphis petition 03-036-02p)event 281-24-236 (USDA aphis petition 03-036-01p); event MON158985 (USDAaphis petition 00-342-01p); event MON531 (USDA aphis petition94-308-01p) or event COT102 (=Syngenta vip3A) USDA aphis petition03-155-01p. Elite event EE GH5 can also be combined with herbicetolerant elite events such as event MON1445 (USDA aphis petition95-045-01p) or event MON88913 (USDA aphis petition 04-086-01p).

As used in the claims below, unless otherwise clearly indicated, theterm “plant” is intended to encompass plant tissues, at any stage ofmaturity, as well as any cells, tissues, or organs taken from or derivedfrom any such plant, including without limitation, any seeds, leaves,stems, flowers, roots, single cells, gametes, cell cultures, tissuecultures or protoplasts.

Reference seed comprising elite event EE-GH5 was deposited as EE-GH5 atthe ATCC (10801 University Blvd., Manassas, Va. 20110-2209) on Jan. 22,2007, under ATCC accession number PTA-8171. An alternative name forEE-GH5 is T304-40

As used in the claims below, unless otherwise clearly indicated, theterm “plant” is intended to encompass plant tissues, at any stage ofmaturity, as well as any cells, tissues, or organs taken from or derivedfrom any such plant, including without limitation, any seeds, leaves,stems, flowers, roots, single cells, gametes, cell cultures, tissuecultures or protoplasts.

The above description of the invention is intended to be illustrativeand not limiting.

Various changes or modifications in the embodiments described may occurto those skilled in the art. These can be made without departing fromthe spirit or scope of the invention.

1-10. (canceled)
 11. A method for identifying elite event EE-GH5 inbiological samples, comprising amplifying a nucleic acid present in saidbiological samples with at least two primers, wherein one of saidprimers recognizes the 5′ or 3′ flanking region of EE-GH5 and the otherprimer recognizes the foreign DNA of EE-GH5 contiguous therewith, said5′ flanking region comprises the nucleotide sequence of SEQ ID No. 1from nucleotide 1 to nucleotide 98 or the nucleotide sequence of SEQ IDNo. 16 from nucleotide 1 to nucleotide 563 and said foreign DNAcontiguous with said 5′ flanking region comprises the complement of SEQID No. 1 from nucleotide 99 to nucleotide 334, said 3′ flanking regioncomprises the nucleotide sequence of the complement of SEQ ID No. 2 fromnucleotide 41 to nucleotide 452 and said foreign DNA contiguous withsaid 3′ flanking region comprises the nucleotide sequence of SEQ ID No.2 from nucleotide 1 to nucleotide
 40. 12. The method of claim 11,wherein said method comprises amplifying a DNA fragment of between 100and 500 bp from a nucleic acid present in said biological samples usinga polymerase chain reaction with said at least two primers.
 13. Themethod of claim 12, wherein said primer recognizing the 5′ flankingregion consist of a nucleotide sequence of 17 to 200 consecutivenucleotides selected from the nucleotide sequence of SEQ ID No 1 fromnucleotide 1 to nucleotide 98 or the nucleotide sequence of SEQ ID No 16from nucleotide 1 to nucleotide 563 or said primer recognizing the 3′flanking region of EE-GH5 consist of a nucleotide sequence of 17 to 200consecutive nucleotides selected from the nucleotide sequence of thecomplement of SEQ ID No 2 from nucleotide 41 to nucleotide 452, and saidprimer recognizing a sequence within the foreign DNA consists of 17 to200 consecutive nucleotides selected from the nucleotide sequence of thecomplement of SEQ ID No. 1 from nucleotide 99 to nucleotide 334 or thenucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 40.14. The method of claim 12, wherein said primer recognizing the 5′flanking region comprises at its extreme 3′ end a nucleotide sequence ofat least 17 consecutive nucleotides selected from the nucleotidesequence of SEQ ID No 1 from nucleotide 1 to nucleotide 98 or thenucleotide sequence of SEQ ID No 16 from nucleotide 1 to nucleotide 563or said primer recognizing the 3′ flanking region of EE-GH5 comprises atits extreme 3′ end a nucleotide sequence of at least 17 consecutivenucleotides selected from the nucleotide sequence of the complement ofSEQ ID No 2 from nucleotide 41 to nucleotide 452, and said primerrecognizing a sequence within the foreign DNA comprises at its 3′ end atleast 17 consecutive nucleotides selected from the nucleotide sequenceof the complement of SEQ ID No. 1 from nucleotide 99 to nucleotide 334or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 tonucleotide
 40. 15. The method of claim 12, wherein said primers comprisethe sequence of SEQ ID No. 3 and SEQ ID No. 6, respectively.
 16. Themethod of claim 12, wherein said method comprises amplifying a fragmentof about 369 bp.
 17. A kit for identifying elite event EE-GH5 inbiological samples, said kit comprising one primer recognizing the 5′flanking region of EE-GH5, said 5′ flanking region having the nucleotidesequence of SEQ ID No 1 from nucleotide 1 to nucleotide 98 or thenucleotide sequence of SEQ ID No 16 from nucleotide 1 to nucleotide 563or one primer recognizing the 3′ flanking region of EE-GH5, said 3′flanking region having the nucleotide sequence of the complement of SEQID No 2 from nucleotide 41 to nucleotide 452 and one primer recognizinga sequence within the foreign DNA, said foreign DNA having thenucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 99to nucleotide 334 or the nucleotide sequence of SEQ ID No 2 fromnucleotide 1 to nucleotide
 40. 18. The kit of claim 17, wherein saidprimer recognizing the 5′ flanking region consists of a nucleotidesequence of 17 to 200 consecutive nucleotides comprising the nucleotidesequence of SEQ ID No 1 from nucleotide 1 to nucleotide 98 or thenucleotide sequence of SEQ ID No 16 from nucleotide 1 to nucleotide 563or said primer recognizing the 3′ flanking region of EE-GH5 consists ofa nucleotide sequence of 17 to 200 consecutive nucleotides of thecomplement of SEQ ID No 2 from nucleotide 41 to nucleotide 452, and saidprimer recognizing a sequence within the foreign DNA consists of 17 to200 consecutive nucleotides comprising the nucleotide sequence of thecomplement of SEQ ID No. 1 from nucleotide 99 to nucleotide 334 or thenucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 40.19. The kit of claim 17, wherein said primer recognizing the 5′ flankingregion comprises at its extreme 3′ end a nucleotide sequence of at least17 consecutive nucleotides selected from the nucleotide sequence of SEQID No 1 from nucleotide 1 to nucleotide 98 or the nucleotide sequence ofSEQ ID No 16 from nucleotide 1 to nucleotide 563 or said primerrecognizing the 3′ flanking region of EE-GH5 comprises at its extreme 3′end a nucleotide sequence of at least 17 consecutive nucleotidesselected from the nucleotide sequence of the complement of SEQ ID No 2from nucleotide 41 to nucleotide 452, and said primer recognizing asequence within the foreign DNA comprises at its 3′ end at least 17consecutive nucleotides selected from the nucleotide sequence of thecomplement of SEQ ID No. 1 from nucleotide 99 to nucleotide 334 or thenucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 40.20. The kit of claim 17, comprising a primer consisting of the sequenceof SEQ ID No. 3 and a primer consisting of the sequence of SEQ ID No. 6.21-25. (canceled)
 26. A method for identifying elite event EE-GH5 inbiological samples comprising hybridizing a nucleic acid of biologicalsamples with a specific probe that recognizes a part of the 5′ or 3′flanking region of EE-GH5 and a part of the foreign DNA of EE-GH5contiguous therewith, said 5′ flanking region comprises the nucleotidesequence of SEQ ID No. 1 from nucleotide 1 to nucleotide 98 or thenucleotide sequence of SEQ ID No. 16 from nucleotide 1 to nucleotide 563and said foreign DNA contiguous with said 5′ flanking region comprisesthe complement of SEQ ID No. 1 from nucleotide 99 to nucleotide 334,said 3′ flanking region comprises the nucleotide sequence of thecomplement of SEQ ID No. 2 from nucleotide 41 to nucleotide 452 and saidforeign DNA contiguous with said 3′ flanking region comprises thenucleotide sequence of SEQ ID No. 2 from nucleotide 1 to nucleotide 40.27. The method of claim 26, wherein the sequence of said specific probehas at least 80% sequence identity with a sequence comprising part ofthe 5′ flanking sequence or the 3′ flanking sequence of EE-GH5 and thesequence of the foreign DNA contiguous therewith.
 28. The method ofclaim 27, wherein the sequence of said specific probe has at least 80%sequence identity with SEQ ID No. 1 from nucleotide 78 to 1 19 or SEQ IDNo. 2 from nucleotide 20 to 61, or the complement of said sequences. 29.A kit for identifying elite event EE-GH5 in biological samples, said kitcomprising a specific probe, capable of hybridizing specifically to partof the 5′ or 3′ flanking region of EE-GH5 and a part of the foreign DNAof EE-GH5 contiguous therewith, said 5′ flanking region comprises thenucleotide sequence of SEQ ID No. 1 from nucleotide 1 to nucleotide 98or the nucleotide sequence of SEQ ID No. 16 from nucleotide 1 tonucleotide 563 and said foreign DNA contiguous with said 5′ flankingregion comprises the complement of SEQ ID No. 1 from nucleotide 99 tonucleotide 334, said 3′ flanking region comprises the nucleotidesequence of the complement of SEQ ID No 2 from nucleotide 41 tonucleotide 452 and said foreign DNA contiguous with said 3′ flankingregion comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide1 to nucleotide
 40. 30. The kit of claim 29, wherein the sequence ofsaid specific probe has at least 80% sequence identity with a sequencecomprising part of the 5′ flanking sequence or the 3′ flanking sequenceof EE-GH5 and the sequence of the foreign DNA contiguous therewith. 31.The kit of claim 30, wherein the sequence of said specific probe has atleast 80% sequence identity with SEQ ID No. 1 SEQ ID No. 1 fromnucleotide 78 to 1 19 or SEQ ID No. 2 from nucleotide 20 to 61, or thecomplement of said sequences.
 32. A specific probe for theidentification of elite event EE-GH5 in biological samples thatrecognizes a part of the 5′ or 3′ flanking region of EE-GH5 and a partof the foreign DNA of EE-GH5 contiguous therewith, said 5′ flankingregion comprises the nucleotide sequence of SEQ ID No. 1 from nucleotide1 to nucleotide 98 or the nucleotide sequence of SEQ ID No. 16 fromnucleotide 1 to nucleotide 563 and said foreign DNA contiguous with said5′ flanking region comprises the complement of SEQ ID No. 1 fromnucleotide 99 to nucleotide 334, said 3′ flanking region comprises thenucleotide sequence of the complement of SEQ ID No 2 from nucleotide 41to nucleotide 452 and said foreign DNA contiguous with said 3′ flankingregion comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide1 to nucleotide
 40. 33. The probe of claim 32, which has at least 80%sequence identity with a sequence comprising part of the 5′ flankingsequence or the 3′ flanking sequence of EE-GH5 and the sequence of theforeign DNA contiguous therewith, or the complement thereof.
 34. Theprobe of claim 33, which has at least 80% sequence identity with SEQ IDNo. 1 from nucleotide 78 to 1 19 or SEQ ID No. 2 from nucleotide 20 to61, or the complement of said sequences.
 35. (canceled)
 36. A method forconfirming seed purity comprising (a) (i) amplifying a nucleic sequencepresent in a seed sample using at least two primers, wherein one of saidprimers recognizes the 5′ or 3′ flanking region of EE-GH5 and the otherprimer recognizes the foreign DNA of EE-GH5 contiguous therewith, or(ii) hybridizing a nucleic sequence present in a seed sample with aspecific probe that recognizes a part of the 5′ or 3′ flanking sequenceof EE-GH5 and a part of the foreign DNA of EE-GH5 contiguous therewith,said 5′ flanking region comprises the nucleotide sequence of SEQ ID No.1 from nucleotide 1 to nucleotide 98 or the nucleotide sequence of SEQID No. 16 from nucleotide 1 to nucleotide 563 and said foreign DNAcontiguous with said 5′ flanking region comprises the complement of SEQID No. 1 from nucleotide 99 to nucleotide 334, said 3′ flanking regioncomprises the nucleotide sequence of the complement of SEQ ID No. 2 fromnucleotide 41 to nucleotide 452 and said foreign DNA contiguous withsaid 3′ flanking region comprises the nucleotide sequence of SEQ ID No.2 from nucleotide 1 to nucleotide 40; and (b) confirming purity of saidseed sample.
 37. A method for screening seeds for the presence of EE-GH5(a) (i) amplifying a nucleic sequence present in a seed sample using atleast two primers, wherein one of said primers recognizes the 5′ or 3′flanking region of EE-GH5 and the other primer recognizes the foreignDNA of EE-GH5 contiguous therewith, or (ii) hybridizing a nucleicsequence present in a seed sample with a specific probe that recognizesa part of the 5′ or 3′ flanking sequence of EE-GH5 and a part of theforeign DNA of EE-GH5 contiguous therewith, said 5′ flanking regioncomprises the nucleotide sequence of SEQ ID No. 1 from nucleotide 1 tonucleotide 98 or the nucleotide sequence of SEQ ID NO: 16 fromnucleotide 1 to nucleotide 563 and said foreign DNA contiguous with said5′ flanking region comprises the complement of SEQ ID No. 1 fromnucleotide 99 to nucleotide 334, said 3′ flanking region comprises thenucleotide sequence of the complement of SEQ ID No 2 from nucleotide 41to nucleotide 452 and said foreign DNA contiguous with said 3′ flankingregion comprises the nucleotide sequence of SEQ ID No. 2 from nucleotide1 to nucleotide 40; and (b) screening said seed sample for the presenceof elite event EE-GH5. 38-39. (canceled)
 40. A method of detecting thepresence of elite event EE-GH5 in biological samples throughhybridization with a substantially complementary labeled nucleic acidprobe in which the probe:target nucleic acid ratio is amplified throughrecycling of the target nucleic acid sequence, said method comprising:a) hybridizing said target nucleic acid sequence to a first nucleic acidoligonucleotide comprising the nucleotide sequence of SEQ ID No 1 fromnucleotide 99 to nucleotide 1 16 or its complement or said first nucleicacid oligonucleotide comprising the nucleotide sequence of SEQ ID No 2from nucleotide 23 to nucleotide 40 or its complement; b) hybridizingsaid target nucleic acid sequence to a second nucleic acidoligonucleotide comprising the nucleotide sequence of SEQ ID No 1 fromnucleotide 81 to nucleotide 98 or its complement or said labeled nucleicacid probe comprising the nucleotide sequence of SEQ ID No 2 fromnucleotide 41 to nucleotide 58 or its complement, wherein said first andsecond oligonucleotide overlap by at least one nucleotide and whereineither said first or said second oligonucleotide is labeled to be saidlabeled nucleic acid probe; c) cleaving only the labeled probe withinthe probe:target nucleic acid sequence duplex with an enzyme whichcauses selective probe cleavage resulting in duplex disassociation,leaving the target sequence intact; d) recycling of the target nucleicacid sequence by repeating steps (a) to (c); and e) detecting cleavedlabeled probe, thereby determining the presence of said target nucleicacid sequence.
 41. An isolated nucleic acid molecule comprising (i) anucleotide sequence essentially similar to SEQ ID No. 1 from nucleotide88 to nucleotide 109 or SEQ ID No. 2 from nucleotide 30 to 50, or thecomplement of said sequences; or (ii) the nucleotide sequence of SEQ IDNo.
 12. 42. The isolated nucleic acid molecule of claim 41 comprising anucleotide sequence essentially similar to SEQ ID No. 1 from nucleotide78 to nucleotide 119 or SEQ ID No. 2 from nucleotide 20 to 60, or thecomplement of said sequences.
 43. (canceled)
 44. The method of claim 11,comprising amplifying a DNA fragment of about 262 bp using primersconsisting essentially of the nucleotides sequences of SEQ ID No 10 andSEQ ID No 11 respectively.
 45. The kit of claim 17, comprising twooligonucleotide primers consisting essentially of the nucleotidessequences of SEQ ID No 10 and SEQ ID No 11 respectively.